Wei Qi, Wang Li, Zhang Yichen, Abulimiti Saidaiguli, Wang Jie, Xu Xingya
Institute of Future Agriculture, Northwest A&F University, Yangling, Shaanxi 712100, People's Republic of China.
College of Enology, Northwest A&F University, Yangling, Shaanxi 712100, People's Republic of China.
G3 (Bethesda). 2025 Jul 9;15(7). doi: 10.1093/g3journal/jkaf111.
Cohesin is a heteropentameric protein complex that holds sister chromatids together from S phase to anaphase. Its 2 structural maintenance of chromosome subunits form a heterodimer, consisting of an ATPase head domain and a hinge domain connected by long coiled coils. Kleisin subunit associates with the head. Here, using Schizosaccharomyces pombe, we genetically dissected cohesin dynamics based on the relationship between the mutations causing temperature-sensitive and their suppressor mutations. First, we identified suppressor mutations that could rescue the lethality caused by cohesin ATPase mutations. Mutations in the DNA-binding domain of cohesin loader Mis4, or in cell-cycle genes encoding MBF transcription factor complex or Wee1 kinase, rescued both Psm1 and Psm3 ATPase mutants. Then, we performed targeted mutagenesis in both ATPase domains for single-amino-acid substitutions that can rescue the lethality of a kleisin ts mutant at restrictive temperature. Comparison of mutations obtained in Psm1 and Psm3 ATPase domains revealed that analogous mutations in the 2 ATPase domains were frequently observed. Last, suppressors of a coiled-coil mutation were mapped in coiled coils, indicating that proper folding of coiled coils is critical for cohesin functions. Suppressors of a hinge interface mutation are frequently located at the other hinge interface, indicating that the 2 cohesin hinge interfaces work collaboratively in hinge-hinge interactions. Overall, genetic dissection of the relationship between cohesin lethal mutations and their suppressor mutations reflects cohesin dynamics in vivo.
黏连蛋白是一种异源五聚体蛋白复合物，从S期到后期都将姐妹染色单体结合在一起。其两个染色体结构维持亚基形成一个异源二聚体，由一个ATP酶头部结构域和一个通过长卷曲螺旋连接的铰链结构域组成。kleisin亚基与头部结合。在这里，我们利用粟酒裂殖酵母，基于导致温度敏感的突变与其抑制突变之间的关系，对黏连蛋白动力学进行了遗传学剖析。首先，我们鉴定出了能够挽救由黏连蛋白ATP酶突变导致的致死性的抑制突变。黏连蛋白装载因子Mis4的DNA结合结构域中的突变，或编码MBF转录因子复合物或Wee1激酶的细胞周期基因中的突变，挽救了Psm1和Psm3 ATP酶突变体。然后，我们在两个ATP酶结构域中进行了靶向诱变，进行单氨基酸替换，以挽救kleisin温度敏感突变体在限制温度下的致死性。对在Psm1和Psm3 ATP酶结构域中获得的突变进行比较发现，在两个ATP酶结构域中经常观察到类似的突变。最后，卷曲螺旋突变的抑制子被定位在卷曲螺旋中，表明卷曲螺旋的正确折叠对黏连蛋白功能至关重要。铰链界面突变的抑制子经常位于另一个铰链界面，表明两个黏连蛋白铰链界面在铰链-铰链相互作用中协同工作。总体而言，对黏连蛋白致死突变与其抑制突变之间关系的遗传学剖析反映了体内黏连蛋白的动力学。
